(1) Field of the Invention
This invention relates generally to the genesis and malignancy of tumors, and more particularly to methods for screening substances for oncogenic activity and to methods for assessing tumor aggressiveness.
(2) Description of the Related Art
Almost a century ago, Paul Erlich suggested that the immune system played a role in the elimination of spontaneously arising tumor cells (Ned. Tiijdschr. Geneeskd. 5(Pt. 1): 273, 1909, incorporated herein by reference). In 1970, Burnet coined the term xe2x80x9cimmune surveillancexe2x80x9d to embody this concept and proposed that T cells would function as the major effectors in this system (Progr. Exp. Tumor Res. 13:1, 1970, incorporated herein by reference).
Recently, insight into the nature of the immune responses to tumors that were not nascently formed was provided by a study which suggested that the cytokine interferonxcex3 (IFNxcex3) plays an important role in promoting tumor cell recognition and elimination (Dighe et al., Immunity 1:447-456, 1994, which is incorporated herein by reference). In this study, tumor cells derived from two methylcholanthrene-induced murine sarcomas in genetically different mice were transfected with a plasmid encoding a cytoplasmically truncated form of the murine IFNxcex3-receptor ligand binding chain (xcex1 chain). These cell lines are completely unresponsive to IFNxcex3 due to overexpression of the functionally inactive IFNxcex3-receptor xcex1 chain at the cell surface. When these IFNxcex3-insensitive tumor cells were inoculated subcutaneously at low amounts (1 to 2xc3x97104 cells/mouse) into normal syngeneic mice, they formed rapidly progressing tumors in at least 80% of the injected mice. In contrast, mice inoculated with the same amount of IFNxcex3 sensitive wild type tumor cells did not develop tumors. Dighe et al concluded that the development of host responses to the tumor studied requires the production of IFNxcex3 by host cells, the capacity of the tumor to respond to IFNxcex3, and the development of specific T cell immunity.
Although this study identified a role for IFNxcex3 in promoting rejection of transplantable tumors, they did not address the critical question of whether IFNxcex3 participates in promoting host responses to nascently forming transformed cells, i.e., whether it is involved in promoting tumor surveillance. More importantly, the study did not address whether IFNxcex3 responsiveness of the host cell played a role in tumor surveillance, which would be of critical importance in the development of an oncogenic screening method.
In another study of the physiological role of IFNxcex3 in the immune response, knockout mice with an inactivated gene for the IFNxcex3 receptor xcex1 chain were made and shown to be viable with no apparent phenotypic anomalies by 12 months (Huang et al., Science 259:1742-1745, 1993, incorporated herein by reference). The immune system in these IFNxcex3Rxe2x88x92/xe2x88x92 mice appeared to develop normally in that no differences in the major lymphocyte subpopulations between mutant and wild-type mice were observed. However, the mutant mice had increased susceptibility to infection by Listeria monocytogenes and vaccinia virus despite normal cytotoxic and T helper cell responses. In addition, while the IFNxcex3-unresponsive mice generated a normal antigen-specific IgM and IgG1 response, they failed to develop a normal IgG2a response as indicated by decreased titers of antigen-specific IgG2a antibodies at twelve days after immunization with antigen.
Increased susceptibility to infection by microbial pathogens and viruses was also observed in knockout mice deficient in STAT1, an IFN-specific cytosolic transcription factor in the JAK-STAT signaling pathway (Meraz, et al., Cell 84:431-442, 1996, incorporated herein by reference). While these STAT1-deficient mice showed no overt developmental abnormalities and had normal populations and subpopulations of T cells, B cells, and macrophages, they died after infection with doses of Listeria monocytogenes and VSV that are sublethal in normal mice. Cells derived from the mutant mice were not responsive to IFNxcex1 and IFNxcex3 but did respond normally to other cytokine ligands, including growth hormone, epidermal growth factor, and interleukin-10.
A recent study examined the susceptibility of perforin-deficient (PKO) mice to tumor induction by a sarcoma-inducing carcinogen, methylcholanthrene (MCA), a papilloma-inducing carcinogen, 7,12-dimethylbenzanthracene (DBMA) plus 12-O-tet-radecanoylphorbol-13-acetate (TPA) (DBMA+TPA), and a sarcoma-inducing virus, Moloney murine sarcoma virus (MoMSV) (van den Broek et al., J. Exp. Med., 184:1781-1790, 1996). PKO mice subcutaneously injected with MCA developed sarcomas at the injection site at a higher frequency and with accelerated onset of tumor than observed in normal MCA-treated mice. In addition, although PKO and normal mice injected intramuscularly with MoMSV displayed similar numbers and kinetics of tumor onset, MoMSV-induced tumors were larger and regression was retarded in the PKO mice than in normal mice. However, the incidence and kinetics of DMBA+TPA-induced papillomas were similar in PKO and normal mice. The authors concluded that several mechanisms probably control tumor growth, with perforin-mediated cytotoxicity playing a role in some types of tumors, but not in others.
Assessing the carcinogenic potential of chemical compounds is indispensable in drug development and in identifying environmental carcinogens. Currently, the gold standard carcinogenicity test is the rodent bioassay performed by the National Toxicology Program (NTP) at Research Triangle Park, N.C. (Ashby et al., Mutat. Res., 257: 229-306, 1991, incorporated herein by reference). The NTP rodent bioassay lasts more than two years, requires a large number of experimental animals, a large amount of laboratory space for animal testing, and a large number of laboratory technicians. The cost of the NTP bioassay is so high that only a few chemicals per year can be evaluated. However, there are many chemicals in commercial use or in the environment that have not been tested, and thousands of new chemicals are synthesized every year. Thus, there is much interest in developing improved animal bioassays that can evaluate the oncogenic potential of chemical compounds within a relatively short period of time.
Recently, in a brief reference to unpublished observations, Bach et al. (Annu. Rev. Immunol. 15:563-591, 1997, incorporated herein by reference) stated that the chemical carcinogen 3-Methylcolanthrene produced more tumors in xcex1 chain knockout mice than in wild-type controls. This reference discussed the role of IFNxcex3 in host surveillance. Nevertheless, the authors did not provide any suggestion as to whether the knockout mice or wild-type controls could be used in a carcinogenicity screening model nor did they provide sufficient details of their findings to allow one to assess the possibility of such use.
Genetically modified mice that carry specific oncogenes or inactivated tumor-suppressor genes have been proposed as candidate animal models for rapid carcinogenicity testing. (See, e.g., Tennant et al., Envir. Health Perspect. 103:942-950, 1995; Yamamoto et al., Carcinogenesis 17:2455-2461, 1996; and Berns, U.S. Pat. No. 5,174,986, each of which is incorporated herein by reference). Mutant mice that reportedly respond more rapidly and at higher frequency to various carcinogens than wild-type mice include v-Ha-ras transgenic mice (Tennant et al., supra), c-Ha-ras transgenic mice (Yamamoto et al., supra), transgenic mice that overexpress the pim-1 oncogene in lymphoid tissues (Storer et al., Carcinogenesis 16:285-293, 1995, incorporated herein by reference and Berns, supra), and p53 heterozygous knockout mice (Tennant et al., supra).
These known animal models have characteristics that may limit their use in a carcinogenic screening assay. For example, the p53xe2x88x92/+ mice apparently respond differently to different classes of carcinogens in that tumors were induced by the mutagenic carcinogens p-cresidine and 4-vinyl-1-cyclohexene diepoxide (VCD), but not by the nonmutagenic carcinogens N-methylolacrylamide (NMOA) and reserpine (Tennant et al., supra). In addition, transgenic mice that express an oncogene in only one tissue may not be able to detect the broadest range of tissue specific carcinogenic activity. Indeed, it was recently suggested that data showing a very weak lymphoma response by pim-1 mice to three genotoxic carcinogens that do not normally induce lymphomas and a negative response to a known mouse lymphomagen raised concern that pim-1 mice are not sufficiently sensitive to established carcinogens to justify their routine use in a short-term carcinogenic screening assay (Storer et al, supra). Finally, data obtained with transgenic mice having inappropriate expression of an oncogene may not be predictive of the effect of such compounds in animals with normal expression of the oncogene. Therefore, it would be desirable to develop a screening assay that uses an animal model in which tumors are rapidly induced by a wide variety of mutagenic and nonmutagenic carcinogens that act on different target tissues and through different mechanisms.
Briefly, therefore, the present invention is directed to a method for rapid in vivo screening of substances for carcinogenic activity and, in addition, a new method for diagnosing the clinical aggressiveness of tumors. Accordingly, the inventors herein have succeeded in discovering that IFNxcex3-insensitive mice are surprisingly much more susceptible to chemically induced tumor formation than IFNxcex3-sensitive, parental mice. Moreover, it has been discovered that a significant number of tumor cell lines are unresponsive or show reduced responsiveness to IFNxcex3.
One aspect of the present invention, therefore, provides a method to determine the oncogenic potential of a substance of interest, which comprises administering the substance to at least one IFNxcex3-insensitive test animal and monitoring the test animal for tumor formation. Tumors occurring at a higher frequency or at an earlier time in the IFNxcex3-insensitive test animal than spontaneously arising tumors in an untreated IFNxcex3-insensitive animal indicates the substance is oncogenic. Reference to an untreated animal is intended to mean a control animal not receiving administration of the test substance or an animal being evaluated during a period in which the test substance has not been administered although the test substance could be given either before or after the period of no treatment. The untreated animal can receive administration of a carrier vehicle in which the drug would have been administered or, alternatively, the untreated animal can receive no administration at all.
The method also embraces administering a known dose of the substance to each member of a group of IFNxcex3 insensitive test animals and comparing the rate and/or frequency of tumors in the test group with that for tumors arising in a control group of IFNxcex3-insensitive animals treated with a known, oncogenic amount of a known carcinogen, thus providing an indication of the oncogenic potential of a test compound relative to that of the known carcinogen.
In another aspect of the invention, a method for predicting tumor aggressiveness is provided. The method comprises testing a tumor sample from a patient for sensitivity to IFNxcex3. Insensitivity to IFNxcex3 would indicate that the tumor is less likely to be recognized by the patient""s immune system and is thus likely have an extremely aggressive clinical course.
Among the several advantages found to be achieved by the present invention, therefore, may be noted the provision of a new in vivo approach for testing the oncogenicity or carcinogenicity of substances in which the method is inexpensive and simple to perform, requiring only a relatively short testing period compared to currently available methods; and the provision of methods for predicting the aggressiveness of a tumor in a patient.